The overall goal of the research proposed in this program project application is to identify the cellular and molecular triggers that initiate fatal cardiac arrhythmias and to determine novel molecular-targeted therapeutic strategies to treat them. The central hypothesis is that sudden cardiac death (SCD) can be caused by disruption of molecular complexes and processes that, in normal hearts, underlie balanced regulation of cellular activity and furthermore that altered cellular calcium homeostasis plays a critical role in triggering the resulting arrhythmic activity. It is thus the fundamental assumption of this program that, to understand the mechanistic basis of the events that underlie SCD, an integrative approach is necessary that identifies molecular defects of ion channel complexes that coordinate the function of intracellular (Project 1) and plasma membrane (sarcolemmal) (Project 2) ion channels as well as interaction with and dysregulation of intracellular calcium regulation (Project 3). This project focuses on the roles of key sarcolemmal ionic currents in the generation of abnormal calcium-dependent electrical instability. Experiments that are proposed combine patch clamp measurement of ion channel activity, and intracellular calcium and sodium concentrations in myocytes isolated from genetically-altered mice. There are two specific aims. The first aim is to determine whether mutations in sarcolemmal Na+ channels linked to SCD contribute to spontaneous diastolic calcium-dependent electrical activity. The principal hypothesis of this aim is that sustained Na+ entry via mutation-enhanced late sodium channel currents promotes abnormal calcium-dependent diastolic activity due to a combination of action potential prolongation, increased SR Ca load and extrusion of intracellular calcium during diastole. The second aim isjo investigate mechanisms whereby sympathetic nervous system activation contributes to arrhythmias that can cause SCD. The hypothesis to be tested is that adrenergic stimulation (AS) increases ITI susceptibility in part by combined effects of sustained INa (INaL), and enhanced SR Ca load and leak, providing a mechanism whereby increased sympathetic nervous system (SNS) activity can be pro-arrhythmogenic in the context of sustained INa (INaL) (e.g. as seen in heart failure (chronic AS) and in patients with AKPQ mutations in Nav1.5 (transient AS). The proposed studies are significant because they may provide a mechanism whereby altered plasmalemmal Na channel activity can cause calcium dependent cardiac arrhythmias may lead to novel therapeutic concepts for anti-arrhythmic therapy.